This project will provide a detailed understanding of the mechanism of plasmid DNA transfer during bacterial conjugation. Bacterial mating is a basic means of genetic exchange that has been described in both gram-positive and gram-negative organisms. In gram-negative bacteria, conjugation systems are encoded by plasmids, and mediate the transfer of plasmid and chromosomal DNA from donor to recipient cell. Bacterial conjugation is significant in clinical medicine because plasmids specify both antibiotic resistance and important virulence traits, and conjugation is the major mechanism for the spread of these plasmids in pathogenic gram-negative bacteria. The broad host range conjugative plasmid RK2 will be used as a model system to study plasmid DNA transfer. The RK2 conjugation system differs from the previously studied F factor of E. coli in several fundamental properties, including host range, sex pili, and mating physiology. We have identified and sequenced the origin of transfer (oriT) of RK2, and have isolated the RK2 relaxation complex which nicks at oriT and initiates the transfer process. The major structural feature of the 112 base pair oriT sequence is a 19 base pair inverted repeat, which may represent the DNA recognition site for the transfer proteins. The specific aims of this proposal are: 1) to determine the minimal DNA sequence required for a functional origin of transfer, 2) to determine the structural requirements for oriT and the role of the inverted repeats, and 3) to characterize the transfer proteins which interact with oriT. These studies will provide basic information on the protein-DNA interactions during plasmid transfer. In addition, the project will involve two practical applications of broad interest to bacterial geneticists: 1) construction of improved broad host range cloning vectors based on RK2, and 2) the development of a general system for RK2-mediated transfer of the bacterial chromosome. These applications will facilitate genetic manipulations in many bacteria which currently lack a convenient genetic system.